The field of molecular phylogenetics is being revolutionized with next‐generation sequencing technologies making it possible to sequence large numbers of genomes for non‐model organisms ushering us into the era of phylogenomics. The current challenge is no longer how to get enough data, but rather how to analyse the data and how to assess the support for the inferred phylogeny. Here, we focus on one of the largest animal groups on the planet – butterflies and moths (order Lepidoptera), whose phylogeny remains unresolved despite several recent phylogenomic studies. In this study, we assess the potential causes and consequences of the conflicting phylogenetic hypotheses. With a dataset consisting of 331 protein‐coding genes and the alignment length over 290,000 base pairs, including 200 taxa representing 83% of lepidopteran superfamilies, we compare phylogenetic hypotheses inferred from amino acid and nucleotide alignments. The resulting two phylogenies are discordant, especially with respect to the placement of the superfamily Gelechioidea, which is likely due to compositional bias and possible other model violations. Furthermore, we employed a series of analyses to dissect our dataset and demonstrate that there is sufficient phylogenetic signal to resolve much – but not all – of the lepidopteran tree of life. The relationships among superfamilies within Ditrysia, the most species rich lepidopteran clade containing 98% of the extant species, remain poorly resolved. We conclude that taxon sampling remains an issue even in phylogenomic analyses and recommend that poorly sampled highly diverse groups, such as Gelechioidea in Lepidoptera, should receive extra attention in the future.

The field of molecular phylogenetics is being revolutionised with next-generation sequencing technologies making it possible to sequence large numbers of genomes for non-model organisms ushering us into the era of phylogenomics. The current challenge is no longer how to get enough data, but rather how to analyse the data and how to assess the support for the inferred phylogeny. We focus on one of the largest animal groups on the planet - butterflies and moths (order Lepidoptera). We clearly demonstrate that there are unresolved issues in the inferred phylogenetic relationships of the major lineages, despite several recent phylogenomic studies of the group. We assess the potential causes and consequences of the conflicting phylogenetic hypotheses. With a dataset consisting of 331 protein-coding genes and the alignment length over 290 000 base pairs, including 200 taxa representing 81% of lepidopteran superfamilies, we compare phylogenetic hypotheses inferred from amino acid and nucleotide alignments. The resulting two phylogenies are discordant, especially with respect to the placement of the superfamily Gelechioidea, which is likely due to compositional bias of both the nucleotide and amino acid sequences. With a series of analyses, we dissect our dataset and demonstrate that there is sufficient phylogenetic signal to resolve much of the lepidopteran tree of life. Overall, the results from the nucleotide alignment are more robust to the various perturbations of the data that we carried out. However, the lack of support for much of the backbone within Ditrysia makes the current butterfly and moth tree of life still unresolved. We conclude that taxon sampling remains an issue even in phylogenomic analyses, and recommend that poorly sampled highly diverse groups, such as Gelechioidea in Lepidoptera, should receive extra attention in the future.

Genetic diversity feeds the evolutionary process and allows populations to adapt to environmental changes. However, we still lack a thorough understanding of why hotspots of genetic diversity are so 'hot'. Here, we analysed the relative contribution of bioclimatic stability and genetic admixture between divergent lineages in shaping spatial patterns of genetic diversity in the common toad Bufo bufo along the Italian peninsula. We combined population genetic, phylogeographic and species distribution modelling (SDM) approaches to map ancestral areas, glacial refugia, and secondary contact zones. We consistently identified three phylogeographic lineages, distributed in northern, central and southern Italy. These lineages expanded from their ancestral areas and established secondary contact zones, before the last interglacial. SDM identified widespread glacial refugia in peninsular Italy, sometimes located under the present-day sea-level. Generalized linear models indicated genetic admixture as the only significant predictor of the levels of population genetic diversity. Our results show that glacial refugia contributed to preserving both levels and patterns of genetic diversity across glacial-interglacial cycles, but not to their formation, and highlight a general principle emerging in Mediterranean species: higher levels of genetic diversity mark populations with substantial contributions from multiple genetic lineages, irrespective of the location of glacial refugia.

Mediterranean peninsulas are major biodiversity hotspots, and cold-adapted species are an important component of this biodiversity. However, cold-adapted species contributed surprisingly little to our knowledge of the intimate links between Quaternary environmental changes, species’ responses to these changes, and current patterns of intraspecific biodiversity. Here, we investigated the genetic structure and evolutionary history of a cold-adapted amphibian, the Alpine newt Ichthyosaura alpestris, within the Italian peninsula. Nuclear and mitochondrial markers consistently identified three distinct genetic lineages, whose divergence dates to the Early Pleistocene (1.9 and 0.8 million years ago). Our results show that the Italian peninsula provided multiple Pleistocene refugia to this cold-adapted species, and suggest that allopatric fragmentation followed by secondary admixture have been key events in the formation of its current pattern of genetic diversity. Indeed, estimates of population genetic diversity clearly identified contact populations as those achieving the highest levels of diversity. Such concordance among cold-adapted and temperate species in terms of processes triggering the formation of regional patterns of genetic diversity provides strong support for the hypothesis that gene exchange between divergent lineages, rather than long-term stability of refugial populations, has been the main step toward the formation of hotspots of intraspecific biodiversity.